Vascular oxidant stress plays a key role in many pathologic states including hyperoxia, inflammation, ischemia, pulmonary and cardiovascular diseases. However, current antioxidant therapies are not effective, in part due to sub-optimal delivery to endothelial cells (EC). Previous studies have shown that the antioxidant enzyme (AOE) catalase (that detoxifies freely diffusing H2O2) conjugated with antibodies to endothelial Cell Adhesion Molecules (CAM) accumulates in EC after intravenous injection and protects animals against acute oxidant lung injury. Unfortunately, anti-CAM/AOE conjugates are eliminated from the blood and degraded in the EC within a few hours, affording only transient effects. In order to solve this problem and to achieve higher therapeutic duration and utility, we propose to load catalase into H2O2-permeable, anti-CAM targeted di-block PEG-PLGA Polymer Nano-Carriers (PNC) that would prolong the AOE circulation and protect from lysosomal proteolysis. Pilot data show that catalase loaded inside stealth anti-CAM/PNC degrades H2O2, binds to and protects EC from oxidant injury, circulates in animals for a prolonged time, and accumulates in the lung vasculature. The goal of this grant is to explore, test and optimize this strategy, pursuing the following Specific Aims: 1. Optimize the design of PNC for AOE delivery to EC. PEG-PLA PNC formulation, composition, size, AOE loading, activity and protease resistance, rate of pH-dependent degradation, coupling of anti-CAM, binding, uptake and metabolism by EC, and protection of EC will be studied and optimized in vitro. 2. Characterize the behavior and targeting of anti-CAM/PNC/AOE in vivo. Their blood clearance, systemic effects, biodistribution, EC binding, pulmonary targeting, optimal administration routes and persistence in lungs will be tested in naive animals and animals with oxidant vascular stress. 3. Evaluate the effects of anti-CAM/PNC/AOE in animals. Mouse models of acute H2O2 generation in the pulmonary vasculature and sub-acute oxidant stress in the lungs (hyperoxia) will be employed to evaluate the degree and duration of protection, and refine regimens of AOE targeting and mechanisms of protection. Completion of these Aims will be a major step towards the long-term goal of our research, which is translation of this promising new technology platform into the clinical domain.